Author: Ganjian, Haleh; Nikokar, Iraj; Tieshayar, Azita; Mostafaei, Ali; Amirmozafari, Nour; Kiani, Sara
Date published: January 1, 2011
Staphylococcus aureus is one of the important human pathogens involved in food-related diseases and a common cause of community-associated infection (1, 2). This organism proliferates in food and releases one or more heat-stable enterotoxins, causing food-borne illnesses (3). S. aureus is the most common cause of infections in hospitalized patients and has been a major concern for well over a century (4). The spectrum of diseases caused by this organism is extremely wide, ranging from superficial infections to deep-seated and systemic infections such as pneumonia, endocarditis, osteomyelitis, and sepsis (5). The treatment of staphylococcal infections has become extremely challenging due to the propensity of the organism to rapidly evolve into antibiotic-resistant strains. Antibiotic resistance is an emerging problem worldwide, and widespread use of antibiotics is likely to be the main reason for the increase in antibiotic resistance (6).
Prevalence of resistant S. aureus, especially methicillinresistant S. aureus (MRSA), is increasing in clinical settings. MRSA is one of the most common causes of nosocomial infections. Methicillin, a semi-synthetic penicillinase-resistant penicillin, was introduced in 1960 for the treatment of penicillinase-producing strains of S. aureus; methicillinresistant strains of S. aureus were identified in 1961 (7). Environmental stresses, including temperature, pH, salts, osmotic pressure, and alkaline and acidic conditions can affect the growth rate and population of bacteria (8). To our knowledge, stress and antimicrobial drug resistance has been studied more extensively in Gram-negative bacteria such as Escherichia coli; however, very little is known about these factors with regard to S. aureus.
The aim of this study was to investigate the effect of environmental stress on the antibiotic susceptibility and protein profile of S. aureus as a prototypical Gram-positive bacterium.
3. Materials and Methods
S. aureus (ATCC 25823) was obtained from the Iranian Research Organization for Science and Technology and grown in trypticase soy broth (TSB; Merck) at 37°C. S. aureus cells in the exponential growth phase were exposed to sub-lethal salt stress by using concentrations ranging from 5% to 35% (wt/vol). The stress-treated cells were then harvested by centrifugation (3,000 xg for 15 min) and were re-cultured on mannitol salt agar (MSA, Merck). Bacteria were re-suspended in a tube containing 0.5ml of saline. To standardize the number of bacteria, 0.5 McFarland standard was prepared by adding 99.5ml of 1% sulfuric acid to 0.5ml of 1.175% BaCl2 solution. Spectrophotometric analysis of the bacterial suspension by using 0.5 McFarland standard revealed that the suspension contained l.5xl08 bacteria per milliliter (CFU/ mL). Cell suspensions containing stressed and normal bacterial cells were plated on Mueller-Hinton agar (MHA; Merck Company) plates using a sterile swab. Susceptibility toll antibiotics was tested by performing the disk diffusion method by using commercial disks (MAST Diagnostics, Merseyside, UK) according to the Clinical Laboratory Standards Institute guidelines (9).
The antibiotics used and their disk potencies were as follows: erythromycin, I5]ig, penicillin G, 1OU; gentamicin, 10]ig; ciprofloxacin, 5]ig, cefalexin, 30]ig, chloramphenicol, 30]ig; co-trimoxazole, 25]ig, rifampicin, 5]ig, clindamycin, 2]ig, cephalothin, 30]ig, and methicillin (25yg). The plates were incubated at 37°C for 17 to 24 h and were then examined 4times for the development of zones of inhibition in each lawn growth around the disc. The pooled protein from stressed and non-stressed (control) bacterial cells were analyzed using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (10). Statistical analyses wereperformed by ANOVA by using Statistical Package for Social Sciences (SPSS, versionll.5). AP value of ^0.05 was considered statistically significant.
S. aureus ATCC 25823 cultures were gradually adapted to the 4 salt concentrations (5.0%, 15%, 25%, and 35.0% [wt/vol]), in various periods ranging from 5 to 20 min. Evaluation of antimicrobial drug resistance pattern revealed significant differences in zone sizes between the test and control suspensions under salt stress. Salt-stressed S. aureus suspensions had significantly smaller inhibition zonesin the presence of rifampicin, penicillin, and methicillin.However, the zone of gentamicin was bigger to those observed for un-stressed control suspensions (Table). The tested bacterial strain had the highest resistance to methicillin (P<0.001), significantly higher than that shown to any other antibiotics tested, especially in 35% concentration of salt. The SDS-PAGE analysis of pooled proteins at 4 conditions, (i) 5.0%, (ii) 15%, (iii) 25%, and (iv) 35.0% (wt/vol) of salt, in comparison with the control, is shown in Figure 1. Comparison of the protein profiles between control and those treated with different salt concentrations showed that salt treatment decreased the intensity of some of the bands (20 kDa, 40 kDa, and 60 kDa) at salt concentrations of 5% to 35%. In contrast, some protein bands (47 kDa, 66 kDa. and >78 kDa) showed increased intensity in the same range of salt concentrations.
Historically, salt has been used both as an additive and preservative in foods, and abundant information on it can be found in the literature, salt has often been incorporated as an antimicrobial agent in meat, meat products, or brine solutions (11). In the present study, we showed that S. aureus could grow gradually at 4 salt conditions, (i) 5. 0%, (ii) 15%, (iii) 25%, and (iv) 35. 0% (wt/vol). This finding is consistent those of some previous reports (12, 13). Osmotolerance in this organism is therefore an interesting topic for several reasons. The intracellular concentration of potassium (K) is high and does not change much on salt stress since osmoprotectants can enter S. aureus cells through various transport mechanisms that are activated or induced by salt stress. S. aureus can also activate some genes and express proteins in response to salt stress (14-16). In the present study, antibiotic susceptibility in S. aureus ATCC 25823 decreased in the presence of sub-lethal concentrations of salt. Asimilar response has been reported in wild-type strains of S. aureus isolated from commercial food kitchens (8).
Genotypic changes may be responsible for the development of hyper-resistant varieties of Staphylococcus. By point mutations, target sites for antibiotic binding can be inactivated and heterogeneous populationcan be generated with increased spontaneous mutation rates (hypermutable strains) (8, 17, 18). Our results show that salt stress causes significantly greater changes in methicillin resistance than in any other antibiotics tested. The mechanism behind the staphylococcal resistance to methicillin is attributable to the expression of a unique penicillin-binding protein, PBP2a, which has a much lower affinity for beta-lactam antibiotics (19). Analysis of protein profile in this study shows changes in the protein, which was attributable to the expression and inhibition of some important proteins.
Exposure of microorganisms to sub-lethal concentrations of salt can induce the expression of stress proteins with a profile similar to that of stress protein expression induced by heat shock. Many environmental stresses can induce the Mar (multiple antibiotic resistances) operon that is known to regulate the expression of a large number of genes, including the efflux pump (the arcAB efflux pump) (8, 20). This suggests that using high concentrations of salt in food preservation can lead to the development of population or subpopulation of S. aureus with decreased susceptibility to antibiotics. Results of this study demonstrated that sub-lethal salt stress could significantly alter the antibiotic resistance and protein profile of S. aureus. We conclude that the increased use of salt in food processing may contribute to the development and dissemination of antibiotic resistance S. aureus as food- borne pathogens.
This article is a compilation of results of MS thesis (Code, 20230507882015) from Azad University of Lahijan, Guilan, Iran. The tests were conducted at the Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Gilan University of Medical Sciences, Langeroud.
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Haleh Ganjian1, Iraj Nikokar2*, Azita Tieshayar2, Ali Mostafaei3, Nour Amirmozafari4, Sara Kiani3
1 Azad University of Lahijan, Guilan, IR Iran
2 Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, Guilan, IR Iran
3 Kermanshah University of Medical Sciences, Kermanshah, IR Iran
4 Tehran University of Medical Sciences, Tehran, IR Iran
* Corresponding author: Iraj Nikokar, Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, P.O. Box: 44715-1361, Langeroud, IR Iran. Tel: +981425237070, Fax: +98-1425237171, Email: Nikokariraj@yahoo.com, Nikokariraj@gums.ac.ir
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